It has been recognized for many years that cyanate readily reacts with certain amino acid side chain functional groups of a peptide [see G. R. Stark, Method In Enzymology 11, 590-594 (1967)].
Urea-containing solutions are commonly used to solubilize proteins. One of the disadvantages of the use of urea is that it can dissociate into cyanic acid. The cyanate thus formed often reacts with the primary amine in the protein to yield a carbamylated derivative. This derivative may have biological and antigenic properties that are different from those of the native protein. As a result that the therapeutic efficacy of a carbamylated protein may be compromised. In addition, irreversible carbamylation of primary amines on proteins or peptides could complicate the purification process and/or reduce the biological activities of therapeutic bioproducts.
At equilibrium, an 8 M urea solution may contain 0.02 M cyanate, according to the following reaction:H2N—CO—NH2CNO−+H++NH3−The nonspecific, pH dependent binding reaction between cyanic acid and protein is called carbamylation:CNO−+amino acid side chains→carbamylated protein.The cyanate reaction with —NH2 side chains is irreversible and has an appreciable rate. Also, slightly acidic conditions promote a rapid reaction of cysteine sulfhydryls with residual cyanate derived from urea. However, the carbamylation of the —SH and —imidazole side chains is reversible in slightly alkaline pH. The carbamylated residues are neutral, so that the proteins become less charged and can be reflected in their ion exchange chromatographic behavior.
Protein carbamylation is a major issue both in vivo and in vitro. Lippincott and Apostol (1999) have shown that hemoglobins had a significant level of carbamylated cysteines as an artifact of protein digestion in the presence of urea. Hasuike et al. (2002) have recently shown that cyanate can induce hemolysis by carbamylation of erythrocytes. Thus, carbamylated hemoglobin serves as a marker of posttranslational protein modification associated with such uremic complications as atherosclerosis. Oimomi et al. (1987) measured the activity of carbamylated insulin and showed that both immunological and biological activities changed. In addition, Crompton et al. (1985) had shown that the carbamylation of lens proteins by cyanate causes conformational changes that lead to cataracts. In vitro, the carbamylation of proteins results in lower protein solubility and biological activity, that can lead to a low purification yield and a difficult purification process.
Different methods to prevent carbamylation have been proposed. Lowering the temperature slows down both the cyanate formation and subsequent carbamylation, but increases the viscosity that can impact downstream processes such as filtration and chromatography. Deionization of urea solution only temporarily removes the cyanate from solution. Lowering the pH to 2 decreases cyanate formation but is unattractive for most proteins. Amine-specific derivatization and deprotection is not a convenient quantitative approach. While these approaches have applications in special circumstance, none can be generally applied in the field.
Since cyanate formation in the urea buffer cannot be prevented under the condition of normal protein purification, an alternative approach would be to remove cyanate as it forms. A search for CNO− scavengers has been reported (e.g. H2N—CH2—CH2—NH2 for insulin, see DiMarchi patent, 1986). A good protection agent is considered to be inexpensive, inert, soluble, and readily removable. It has to provide high level of protection, and possibly form irreversible complexes with HOCN at neutral pH. Scavenger design is difficult because each functional group has different reactivity, and the protection mechanism is not clear. Good candidates could be amines that are more reactive with CNO− than the primary amine groups of proteins, and have more than one functional group that are sterically unhindered for faster kinetics.
Two alternatives which can be considered for diminishing losses due to carbamylation are addition of a protective scavenger and reversible amine protection. While the latter approach may permit complete protection it is not always desirable due primarily to the difficulty in achieving quantitative amine-specific derivatization and deprotection. If an appropriate reaction scavenger is available, its utilization is a more desirable and less expensive and demanding alternative to reversible protection. An ideal scavenger, of course, is one that is inexpensive and that provides complete protection from modification while otherwise being totally inert to all other reaction components. Since proteins contain a wide range and diversity of functional groups, each of which possesses a different reactivity toward a particular reagent, it is difficult a priori to predict an effective scavenger. While carbamylation is most rapid at sulfhydryl and imidazole sites, the resulting reaction products are of little concern due to their rapid reversal in slightly alkaline buffers. Modification of peptidyl primary amines (for example, NH2-terminus and lysine residues) occurs at an appreciable rate and, for all practical purposes, is irreversible [see G. R. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235,-3177-3181 (1960)]. At each site of primary amine carbamylation the peptide is reduced in physiological buffers one positive charge, thereby often resulting in diminished peptidyl solubility and/or biological activity. Since cyanate is an equilibrium product of aqueous urea solutions [see J. R. Marier and D. Rose, Anal. Biochem. 7, 304-314 (1964)], all peptides containing reactive functional groups, when handled in the presence of urea, are susceptible to irreversible carbamylation. Urea, being an excellent peptidyl solvent due to its ability to disaggregate structural order, facilitates carbamylation. These undesirable derivatized forms not only represent immediate losses in yield but also constitute complications in purification processes.
To diminish carbamylation of peptides in many prior art urea solutions, the solutions were freed of cyanate prior about immediately prior to use, and all chemical manipulations were conducted at reduced temperatures.
There are several prior art methods and/or processes for the removal of cyanate from mediums and/or solutions. One process, the removal of cyanate by deionization, or through pH reduction to below 2.0, is at best temporary, since ammonium cyanate reappears as an equilibrium product of aqueous urea. As well, the low temperature operational restriction results in slower chemical reactions and complicated operations.
The DiMarchi process, and especially the '513 patent, teaches that if an effective scavenger is to be found, it is important first to determine the optimum conditions for carbamylation. In fact, it was the DiMarchi process that aided in the understanding that there are more than one reactive species when considering the carbamylation of proteins during synthesis. It is commonly understood in the art that if cyanate is the reactive species, the rate of reaction should increase with increasing pH until a limit is reached at a pH slightly above the pKa of the amine. However, if cyanic acid is the reactive entity, the relative rate of reaction with an amine should be biphasic with a pH optimum of approximately 6.5. DiMarchi further taught that an ideal reagent for use in preventing peptide carbamylation (scavenge) during synthesis would have the characteristics of being inert to peptides, capable of forming irreversible complexes with cyanic acid at approximately pH 6.5.±2.0, and be a 1,2-ethylene diamine-like compounds or a compound structurally related to 1,2-ethylene diamine and having some carbamylation inhibition and/or reduction characteristics similar thereto. The DiMarchi process defines a compound structurally related to 1,2-ethylene diamine like compound as:
In which R1, R2, R3, and R4 are groups which, as a composite, do not exert significant changes (1) in the pKa values and (2) in the steric accessibility of the respective amino acid groups relative to the properties of 1,2-ethylene diamine itself. DiMarchi stresses that it is the steric arrangement of the 1,2-ethylene diamine like compound that provide for the scavenging ability.
However, while the 1,2-ethylene diamine like compounds possess good cyanate scavenging ability, they are highly basic and strongly influence pH and buffering capacity when used at the concentration suggested by DiMarchi. Therefore the artfield is in search of other compounds and/or groups of compounds that function as carbamylation inhibitors without this disadvantage of the 1,2-ethylene diamine like compounds. Such compounds should either be much more effective scavengers than 1,2-etylene diamine, so that they can be used in sub-milimolar concentration, or significantly less basic than 1,2-ethylene diamine, preferably with low or no net charge at the experimental conditions, having low impact on the buffering capacity of typical biological buffers when used at a milimolar concentrations. Alternatively, such compounds would have a buffering capacity within or close to the neutral range and could be used as buffers.